Direct regulating effects of transforming growth factor beta on the Leydig cell steroidogenesis in primary culture

The effect of transforming growth factor beta on testicular steroidogenesis was studied by using a model of immature porcine Leydig cells cultured in a chemically defined medium. Leydig cells were cultured in the presence of human or porcine purified TGF beta and the following parameters were measured: cell proliferation, LH/hCG binding, and hCG-stimulated steroid hormone productions (DHEA, DHEAS and testosterone). Whereas TGF beta from the two sources had no effect on Leydig cell multiplication, it markedly inhibited LH/hCG-stimulated DHEA and DHEAS in a time- and dose-dependent manner. The maximal inhibitory effect of this peptide on LH/hCG binding (65% decrease), hCG-stimulated DHEA (77% decrease) and DHEAS (92% decrease) productions was observed with 2 ng/ml for 48 h of treatment. In contrast, TGF beta exerted a biphasic effect on hCG-stimulated testosterone production: stimulating (110% increase) until 2 ng/ml and inhibiting (35% decrease) for higher concentrations. [125I]TGF beta was cross-linked to Leydig cells using disuccinimidyl suberate; cells affinity labelled with [125I]TGF beta exhibit a major labelled band of approx 280 kDa, which has the properties expected from a TGF beta receptor. These data demonstrate that TGF beta is a direct potent regulator of Leydig cell steroidogenic function and its effects are probably mediated via a specific receptor.     

Comparison of the biological actions of TGF beta-1 and TGF beta-2: differential activity in endothelial cells

Beta transforming growth factor (TGF beta) has multiple in vitro biological effects including stimulation or inhibition of proliferation of specific cell types. A second major form of TGF beta, TGF beta-2, has recently been isolated from porcine platelets, from bovine bone matrix, and from several other sources. The two forms of TGF beta are biologically equipotent with the exception that TGF beta-2 was much less active than TGF beta-1 for inhibition of proliferation of a rat pleuripotent hematopoietic stem cell line. During the purification of beta TGF from bone, we obtained two fraction pools that differed in their ability to inhibit 3H-thymidine incorporation into aortic endothelial cells (AEC). We therefore compared highly purified TGF beta-1 and TGF beta-2 isolated from porcine platelets for inhibition of DNA synthesis in mink lung epithelial cells (MvILu), and in AEC, and for stimulation of 3H-thymidine incorporation in calvarial bone cells (CBC) in 3 experiments. TGF beta-1 and TGF beta-2 inhibited cell proliferation in MvILu with no significant differences in the ED50 (31 +/- 8 pg/ml vs 23 +/- 7). TGF beta-2 was much less potent than TGF beta-1 in inhibiting DNA synthesis in AEC (6310 +/- 985 pg/ml vs 101 +/- 34). The reduced specific activity of TGF beta-2 was also observed in adrenal capillary endothelial cells. Both beta-1 and beta-2 stimulated proliferation of CBC (ED50 26 +/- 2 pg/ml vs 10 +/- 4). We also examined the specificity of the MvILu and AEC inhibition assays. Epidermal growth factor (EGF), platelet derived growth factor (PDGF), acidic and basic fibroblast growth factors (FGF), skeletal growth factor (SGF)/insulin-like growth factor-II (IGF-II), and insulin-like growth factor-I (IGF-I) did not inhibit DNA synthesis in either assay system. However, when the growth factors were added to maximal inhibiting concentrations of TGF beta-1, both acidic and basic FGF significantly reduced TGF beta-1 inhibition in AEC. We conclude that (1) inhibition of DNA synthesis in endothelial cells is relatively specific for TGF beta-1, (2) inhibition of DNA synthesis in MvILu is a sensitive and specific assay for generic TGF beta activity but does not distinguish beta-1 from beta-2, (3) the relative inhibition of DNA synthesis in MvILu and AEC may provide a means to quantitatively estimate TGF beta-1 and TGF beta-2, and (4) both TGF beta-1 and TGF beta-2 are potent mitogens for chicken embryonic calvarial bone cells.     

Effects of insulin and somatomedin C on the function of cultured Leydig cells

Porcine cultured Leydig cells (LC) lose hCG receptors and hCG responsiveness (cAMP and testosterone) when they are cultured for three days in a defined medium without insulin or somatomedin C (Sm-C) (Insulin-like growth factor I). In the presence of insulin (50 ng/ml) or of Sm-C (10 ng/ml) the loss of the hCG receptor number and the decreased cAMP response to hCG were prevented, but the steroidogenic response to hCG was only partially prevented. This parameter became normal when cells were pretreated with either Sm-C (10 ng/ml) plus insulin (50 ng/ml) or with insulin alone at high concentrations (5 micrograms/ml). These results indicate that both Sm-C and insulin acting through their own receptors increase Leydig cell steroidogenic capacity by increasing hCG receptor number and improving some step beyond cAMP formation.     

Type I IGF receptors of Leydig cells are upregulated by human chorionic gonadotropin

The effects of human chorionic gonadotropin (hCG) on type I insulin-like growth factor (IGF) receptors of purified Leydig cells were investigated. Sprague-Dawley rats (50 day-old) were treated with a single injection of hCG 10 units intraperitoneally, type I IGF receptors were then determined daily for 4 days. HCG caused a rapid increase in type I IGF receptors within 24 h, which returned to basal by 72 h. There was no significant change in binding affinity. Our present study indicates that type I IGF receptors of Leydig cells are up regulated by hCG, and this may be one mechanism by which hCG and IGF-I interact to enhance Leydig cell steroidogenesis.     

Paracrine regulation of testicular function

Data from several experimental approaches have been reviewed and the findings clearly indicate the existence of multiple interactions between testicular cells and the potential role of these interactions in the paracrine control of testicular functions. Both testicular interstitial fluid and spent media from cultured Sertoli cells had an acute steroidogenic effect on Leydig cells, and this effect is not species specific. The secretion of this steroidogenic factor(s), which is probably a protein, is enhanced by previous FSH treatment of Sertoli cells. Coculture for 2-3 days of pig Leydig cells with homologous or heterologous Sertoli cells enhances Leydig cell specific functions (hCG receptor number and hCG responsiveness) and induces Leydig cell hypertrophy. A similar but less pronounced trophic effect is seen when Leydig cells are cultured with spent media from Sertoli cells cultured in the presence of FSH and high concentrations of insulin, but the spent media from Sertoli cells cultured in the absence of these two hormones inhibits Leydig cell specific functions. Somatomedin-C might play an important role in the positive trophic effect of Sertoli cells on Leydig cells, since this peptide is secreted by Sertoli cells and it has trophic effects on the specific function of Leydig cells. Moreover, Sertoli cells, probably through a diffusible factor and cell-to-cell contacts, control the multiplication, meiotic reduction and maturation of germ cells. In turn, the activity of Sertoli cells is modulated by the stage of neighbouring germ cells. Thus, if a normal Sertoli cell function (which depends not only on FSH but also on Leydig and myoid cell secretory products) is an absolute requirement for germ cell multiplication and maturation, these cells, in turn, cyclically regulate Sertoli cell function and through these cells the size and probably the function of Leydig cells.     

Inhibition of testosterone production by propylthiouracil in rat Leydig cells

Propylthiouracil (PTU) is a thioamide drug used clinically to inhibit thyroid hormone production. However, PTU is associated with some side effects in different organs. In the present study, the acute and direct effects of PTU on testosterone production in rat Leydig cells were investigated. Leydig cells were isolated from rat testes, and an investigation was performed on the effects of PTU on basal and evoked-testosterone release, the functions of steroidogenic enzymes, including protein expression of cytochrome P450 side-chain cleavage enzyme (P450(scc)) and mRNA expression of the steroidogenic acute regulatory protein (StAR). Rat Leydig cells were challenged with hCG, forskolin, and 8-bromo-cAMP to stimulate testosterone release. PTU inhibited both basal and evoked-testosterone release. To study the effects of PTU on steroidogenesis, steroidogenic precursor-stimulated testosterone release was examined. PTU inhibited pregnenolone production (i.e., it diminished the function of P450(scc) in Leydig cells). In addition to inhibiting hormone secretion, PTU also regulated steroidogenesis by diminishing mRNA expression of StAR. These results suggest that PTU acts directly on rat Leydig cells to diminish testosterone production by inhibiting P450(scc) function and StAR expression.     

Effects of growth factors on an intestinal epithelial cell line: transforming growth factor beta inhibits proliferation and stimulates differentiation

The effects of epidermal growth factor transforming growth factor beta (TGF beta) and other growth factors on the proliferation and differentiation of a cell line derived from rat intestinal crypt epithelium (IEC-6) were defined. Incorporation of [3H]-thymidine was stimulated 1.4-2.4 fold by insulin, insulin like growth factor (IGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF) and 2% fetal calf serum (FCS) respectively. Additive stimulation was observed when FCS was supplemented by insulin,IGF-I or PDGF but not EGF. Incorporation of [3H]-thymidine by IEC-6 was strongly inhibited by TGF beta with greater than 80% inhibition of incorporation at concentration approximately equal to 2.0 pM. IEC-6 cells bound 4.1 +/- 0.15 X 10(4) molecules TGF beta/cell and appeared to have only a single class of high affinity receptors (Kd approximately equal to 0.5 pM). TGF beta inhibition was unaffected by the presence of insulin or IGF-I suggesting it inhibits proliferation at a step subsequent to that at which these growth factors stimulate [3H]-thymidine incorporation. TGF beta also reduced the stimulation induced by FCS by 65%. In contrast EGF reduced TGF beta inhibition by 60%. IEC-6 cells demonstrated the appearance of sucrase activity after greater than 18 hours treatment with TGF beta. These findings suggest that TGF beta may inhibit proliferative activity and promote the development of differentiated function in intestinal epithelial cells.     

Somatomedin C/insulin-like growth factor 1: an intratesticular differentiative factor of Leydig cells?

By using immature porcine Sertoli cells cultured in serum-free defined medium, we report that medium conditioned by Sertoli cells contained immunoreactive somatomedin C/insulin-like growth factor 1 (SmC/IGF1) measured following acidic gel filtration. The release of this immunoreactive SmC/IGF1 was slightly increased following Sertoli cell treatment with fibroblast growth factor but not with follicle-stimulating hormone or growth hormone. On the other hand, human biosynthetic SmC/IGF1 exerts a potent stimulatory effect on Leydig cell differentiated functions such as LH/hCG-binding (greater than 4-fold) and hCG-stimulated testosterone secretion (greater than 15-fold). This effect was dose and time dependent and the maximal increase of Leydig cell function was observed following 48 h treatment with 50 ng/ml SmC/IGF1. The steroidogenic action of the peptide was not related to Leydig cell growth since both cell number and 3H-thymidine incorporation into DNA were not or slightly (approximately equal to 1.5-fold) increased in the optimal conditions with SmC/IGF1 treatment (100 ng/ml for 48 h). Moreover, the concomitant treatment of Leydig cells by both arabinoside C (10(-5) M), a DNA synthesis inhibitor, and SmC/IGF1 did not modify the stimulating effect of the peptide on LH/hCG-binding and hCG-stimulated testosterone production. Taken together, the present findings support the concept that Sertoli cell derived SmC/IGF1 could be a potent regulator of Leydig cell differentiated functions.     

Growth regulation of human breast carcinoma occurs through regulated growth factor secretion

We describe studies on human breast cancer in which it is shown that specific growth factors (IGF-I, TGF alpha, PDGF) are secreted by human breast cancer cells and likely to be involved in tumor growth and progression. These activities are regulated by estradiol in hormone-dependent breast cancer and secreted constitutively by hormone-independent cells. These growth factor activities can induce the growth of hormone-dependent cells in vivo in athymic nude mice. Hormone-dependent breast cancer cells also secrete TGF beta, a growth-inhibitory substance, when treated with antiestrogens. TGF beta functions as a negative autocrine growth regulator and is responsible for some of the growth-inhibitory effects of antiestrogens.     

Corticotropin-releasing factor receptors and actions in rat Leydig cells

Rat Leydig cells possess functional high affinity receptors for corticotropin-releasing factor (CRF). CRF inhibited human chorionic gonadotropin (hCG)-induced androgen production in cultured fetal and adult Leydig cells in a dose-dependent manner, but it had no effect on basal testosterone secretion. Comparable inhibitory effects of CRF were observed in the presence or absence of 3-isobutyl-1-methylxanthine. CRF treatment caused a marked reduction of steroid precursors of the androgen pathway (from pregnenolone to testosterone) during gonadotropin stimulation, but it did not influence their basal levels. The inhibitory action of CRF on hCG-induced steroidogenesis was fully reversed by 8-bromo-cAMP but was not affected by pertussis toxin. The action of CRF was rapid; and it was blocked by coincubation with anti-CRF antibody. CRF caused no changes in hCG binding to Leydig cells, and in contrast to other target tissues, CRF did not stimulate cAMP production, indicating that CRF receptors are not coupled to Gs in Leydig cells. These studies have demonstrated that CRF-induced inhibition of the acute steroidogenic action of hCG is exerted at sites related to receptor/cyclase coupling or cAMP formation. The inhibitory effects of CRF in the Leydig cell do not occur through the Gi unit of adenylate cyclase, but could involve pertussis toxin-insensitive G protein(s). These observations demonstrate that CRF has a novel and potent antireproductive effect at the testicular level. Since CRF is synthesized in the testis and is present in Leydig cells, it is likely that locally produced CRF could exert negative autocrine modulation on the stimulatory action of luteinizing hormone on Leydig cell function.     

Differentiation of the adult Leydig cell population in the postnatal testis

Five main cell types are present in the Leydig cell lineage, namely the mesenchymal precursor cells, progenitor cells, newly formed adult Leydig cells, immature Leydig cells, and mature Leydig cells. Peritubular mesenchymal cells are the precursors to Leydig cells at the onset of Leydig cell differentiation in the prepubertal rat as well as in the adult rat during repopulation of the testis interstitium after ethane dimethane sulfonate (EDS) treatment. Leydig cell differentiation cannot be viewed as a simple process with two distinct phases as previously reported, simply because precursor cell differentiation and Leydig cell mitosis occur concurrently. During development, mesenchymal and Leydig cell numbers increase linearly with an approximate ratio of 1:2, respectively. The onset of precursor cell differentiation into progenitor cells is independent of LH; however, LH is essential for the later stages in the Leydig cell lineage to induce cell proliferation, hypertrophy, and establish the full organelle complement required for the steroidogenic function. Testosterone and estrogen are inhibitory to the onset of precursor cell differentiation, and these hormones produced by the mature Leydig cells may be of importance to inhibit further differentiation of precursor cells to Leydig cells in the adult testis to maintain a constant number of Leydig cells. Once the progenitor cells are formed, androgens are essential for the progenitor cells to differentiate into mature adult Leydig cells. Although early studies have suggested that FSH is required for the differentiation of Leydig cells, more recent studies have shown that FSH is not required in this process. Anti-Müllerian hormone has been suggested as a negative regulator in Leydig cell differentiation, and this concept needs to be further explored to confirm its validity. Insulin-like growth factor I (IGF-I) induces proliferation of immature Leydig cells and is associated with the promotion of the maturation of the immature Leydig cells into mature adult Leydig cells. Transforming growth factor alpha (TGFalpha) is a mitogen for mesenchymal precursor cells. Moreover, both TGFalpha and TGFbeta (to a lesser extent than TGFalpha) stimulate mitosis in Leydig cells in the presence of LH (or hCG). Platelet-derived growth factor-A is an essential factor for the differentiation of adult Leydig cells; however, details of its participation are still not known. Some cytokines secreted by the testicular macrophages are mitogenic to Leydig cells. Moreover, retarded or absence of Leydig cell development has been observed in experimental models with impaired macrophage function. Thyroid hormone is critical to trigger the onset of mesenchymal precursor cell differentiation into Leydig progenitor cells, proliferation of mesenchymal precursors, acceleration of the differentiation of mesenchymal cells into Leydig cell progenitors, and enhance the proliferation of newly formed Leydig cells in the neonatal and EDS-treated adult rat testes.     

Primary cultures of Leydig cells from rat, mouse and pig: advantages of porcine cells for the study of gonadotropin regulation of Leydig cell function

Primary cultures of interstitial cells were prepared from the testis of mice, rats, and pigs. The cells were grown in a defined medium supplemented with low (0.1%) serum and insulin, transferrin and epidermal growth factor. Comparisons of the interstitial cell cultures from the three species were made for plating efficiency, cell survival, maintenance of hCG receptors and maintenance of steroidogenic responsiveness to hCG. The porcine cultures had a higher plating efficiency and higher hCG receptor levels per cell than Leydig cells from either rodent. Additionally, the porcine cells showed an increase in testosterone (T) production with hCG stimulation throughout their lifespan in culture while the rodent cultures showed a decrease in T stimulation with time with no stimulation by day 6 in culture. These data indicate that species differences exist in hCG receptor concentrations per cell, the maintenance of hCG receptors and steroidogenic response in culture. The initial high survival, purity and continued functional response of porcine interstitial cell cultures make them a superior system for the study of gonadotropin regulation of Leydig cell function.     

Multiple regulation of testicular steroidogenesis

One single injection of ethylene dimethane sulfonate (EDS) to mature rats causes specific degeneration of testicular Leydig cells which is complete after 3 days. At this time no steroidogenic activities can be detected, indicating that Leydig cells are the source of steroids. The mechanism of this cytotoxic effect of EDS has been investigated with isolated cells. Extensive protein alkylation has been shown to occur in Leydig cells, Sertoli cells and hepatocytes. Steroid production by Leydig cells is always inhibited by EDS, but cytotoxic effects of EDS could only be demonstrated in Leydig cells from mature rats or tumour tissue and not in Leydig cells from immature rats. A new population of Leydig cells develops during the next 2-5 weeks after EDS treatment. In hypophysectomized rats this repopulation only occurs when hCG is given daily. FSH has no effects. The proliferative activity in the interstitial tissue increases within 2 days after administration of hCG or EDS and there are indications that LH and locally produced factors are involved in the proliferation of Leydig cells or Leydig cell precursor cells. Inhibition of cAMP production with inhibitors of adenylate cyclase results in an enhancement of the LH-stimulated steroid production similar to that observed with an LHRH agonist and phospholipase C (PLC). Since the effects of LHRH and PLC on protein phosphorylation and steroid production are similar and different from LH or active phorbol esters, it is proposed that LHRH and PLC may stimulate steroid production via liberation of calcium from a specific intracellular pool. Sterol carrier protein2 (SCP2) which is specifically localized in Leydig cells and regulated by LH probably plays a role in the delivery of cholesterol to the mitochondria although the mechanism of this carrier function is not clear. The results indicate that regulation of Leydig cell development and the steroidogenic activities by gonadotrophins and locally produced factors occur via different transducing systems and regulatory pathways.     

Transforming growth factor beta-1 positively modulates the bioactivity of fibroblast growth factor on corneal endothelial cells

Transforming growth factor beta-1 (TGF beta-1), known as an inhibitor of vascular endothelial cell proliferation in vitro, stimulates bovine corneal endothelial cells (BCE) proliferation. It also positively modulates the response of BCE cells to fibroblast growth factor (FGF) and epidermal growth factor (EGF). This effect is concentration dependent within a physiological range of TGF beta-1, but it is blocked if cells are cultured on extracellular-matrix-coated dishes instead of plastic. TGF beta-1 does not modify the number or the affinity of bFGF receptors on BCE cell surface but increases the bFGF content of these cells. This suggests that TGF beta-1 might act through regulation of bFGF synthesis in BCE cells.     

In vitro interactions between Sertoli cells and steroidogenic cells

Both the cell and the species specificities of the steroidogenic potentiating activity (SPA) of Sertoli cells on Leydig cells were studied using a coculture system. Coculture of purified pig Leydig cells with rat or pig Sertoli cells in the presence of FSH led in both cases, to a significant increase in hCG receptor number and in hCG-stimulated testosterone production. Similarly, coculture of bovine adrenal cells with rat or pig Sertoli cells enhanced the steroidogenic response of adrenal cells to ACTH and angiotensin II. Such effects were not observed when pig Leydig cells or bovine adrenal cells were cocultured with bovine aortic endothelial cells. Coculture of Sertoli and Leydig cells in the presence of hCG, resulted in a significant increase in FSH receptor number and in FSH-induced plasminogen activator activity. Such effects did not occur when Sertoli cells were cocultured with either adrenal or aortic endothelial cells.     

Regulation of gonadotropin receptors, gonadotropin responsiveness, and cell multiplication by somatomedin-C and insulin in cultured pig Leydig cells

We have investigated the effects of insulin and somatomedin-C/insulinlike growth factor I(Sm-C) in purified porcine Leydig cells in vitro on gonadotrophins (hCG) receptor number, hCG responsiveness (cAMP and testosterone production), and thymidine incorporation into DNA. Leydig cells cultured in a serum-free medium containing transferrin, vitamin E, and insulin (5 micrograms/ml) maintained fairly constant both hCG receptors and hCG responsiveness. When they were cultured for 3 days in the same medium without insulin, there was a dramatic decline (more than 80%) in both hCG receptor number and hCG responsiveness. However the cAMP but not the testosterone response to forskolin was normal. Both insulin and Sm-C at nanomolar concentrations prevent the decline of both hCG receptors and hCG-induced cAMP production. This effect of both peptides was dose dependent with an ED50 of about 1 ng/ml and 5 ng/ml for SM-C and insulin, respectively. Insulin and Sm-C had no additive effect on these parameters. At nanomolar concentrations, Sm-C and insulin enhanced hCG-induced testosterone production but the effect of Sm-C was significantly higher than that of insulin. However, the effect of insulin at higher concentrations (5 micrograms/ml) was significantly higher than that of Sm-C at 50 ng/ml. In contrast, at nanomolar concentrations only Sm-C stimulated [3H]-thymidine incorporation into DNA and cell multiplication, the stimulatory effect of insulin on these parameters, was seen only at micromolar concentrations. These results indicate that both Sm-C and insulin acting through their own receptors increase Leydig cell steroidogenic responsiveness to hCG by increasing hCG receptor number and improving some step beyond cAMP formation. In contrast, the mitogenic effects of insulin are mediated only through Sm-C receptors.     

Regulation of gonadotropin receptors and gonadotropin responses in a clonal strain of Leydig tumor cells by epidermal growth factor

The MA-10 line is a clonal strain of Leydig tumor cells that has receptors for human choriogonadotropin (hCG) and mouse epidermal growth factor (mEGF). These cells respond to hCG, cholera toxin, and 8-Br-adenosine 3':5'-monophosphate with increased steroid production. It is reported herein that exposure of the MA-10 cells to mEGF results in a substantial (80 to 90%) reduction in the number of hCG receptors per cell. The loss of hCG receptors is accompanied by a corresponding reduction in the ability of hCG to stimulate steroidogenesis. The steroidogenic responses to cholera toxin and 8-Br-adenosine 3':5'-monophosphate, however, are not affected. Other results presented show that mEGF is not a mitogen for these cells.